1. Field of the Invention
This invention relates to a system used to detect and assay genes, making use of a probe substrate, in particular, a nucleic-acid probe substrate. More particularly, this invention relates to a system having, when gene DNA is detected and assayed by hybridization, the function to achieve contact of a specimen with each nucleic-acid probe under the desired temperature conditions, in respect to a substrate onto which plural kinds of single-stranded nucleic acids have been immobilized in an array as nucleic-acid probes, and a method of detecting and assaying gene nucleic acids by using this system.
2. Related Background Art
As progress of methods for detecting and assaying gene DNA according to hybridization, researches relating to genetic chips (DNA chips, microarrays) are being rapidly advanced which have a use to make a plurality of detection tests simultaneously on gene DNA contained in a specimen, by immobilizing a plurality of nucleic-acid probes on a substrate. Such methods for detecting and assaying gene DNA contained in a specimen, utilizing genetic chips (DNA chips, microarrays) are expected to be applicable to various fields such as molecular biological researches and diagnoses on genetic diseases and infectious diseases.
The basic form of a genetic chip is that, in order to detect a target gene DNA according to hybridization, plural kinds of single-stranded nucleic acids having a complementary base sequence in respect to the base sequence of the target genes have been immobilized in an array on the surface of a substrate made of glass or the like. As the fragments of single-stranded nucleic acids having a complementary base sequence in respect to the base sequence of the target genes, which are utilized as hybridization probes, DNA oligomers called oligo DNA which are synthesized by a chemical route or complementary-stranded DNA fragments called cDNA which are biosynthesized by an enzymatic route using as a template any genes derived from biological tissues are commonly utilized. With regard to the immobilization of fragments of single-stranded nucleic acids to the substrate surface, it is, in the case of the oligo DNA, roughly classified into i) a method in which, like the method disclosed in, e.g., U.S. Pat. No. 5,474,796 (or Japanese Patent Application Laid-Open No. 9-500568; applicant: ProtoGene Laboratories), terminals are previously immobilized and then DNA molecules themselves are one by one synthesized on the substrate to form immobilized nucleic-acid fragments, and ii) a method in which, like the method disclosed in, e.g., Japanese Patent Application Laid-Open No. 11-187900 (applicant: CANON KABUSHIKI KAISHA), oligo DNA is separately synthesized and thereafter nucleic-acid fragments are immobilized to the substrate surface by various binding means.
As means for immobilizing to the substrate surface the nucleic-acid fragments prepared separately, various methods are proposed as exemplified by adsorption immobilization which utilizes electric charges the substrate has and electric charges the nucleic-acid fragments have, and immobilization in which the substrate surface is coated with poly-L-lysine or an aminosilane coupling agent and the resulting coating film is utilized so as to improve immobilization efficiency.
Meanwhile, in cross-hybridization (hybridization reaction) on nucleic-acid probes having been immobilized to the substrate surface, gene DNA contained in a specimen solution is single-stranded and then bound to portions which are complementary to the base sequence of the nucleic-acid probes to form a double-stranded structure. Accordingly, the nucleic-acid probes and the specimen solution must be heated to a stated temperature. In addition, gene DNA not having any complementary base sequence in respect to the base sequence of the nucleic-acid probes but having a similar base sequence also causes, in some cases, pseudo-cross-hybridization (mismatch hybridization reaction) to form gentle bonds to the nucleic-acid probes. Furthermore, unreacted adulterant DNA having no similarity in base sequence may also adhere to the substrate surface by physical adsorption or the like. In order to selectively remove such unnecessary DNA molecules standing bound weakly, they are heated to a temperature at which any redissociation of gene DNA having achieved the intended hybridization reaction does not take place but the detachment of the unnecessary DNA molecules standing bound weakly proceeds, and then washed to remove them.
In detection operation making use of the genetic chip, in order to detect only the target gene DNA in a high reproducibility, it is particularly necessary to control in a high precision the heating temperature in the step of hybridization reaction of the target genes in a specimen with the nucleic acids (probes) having been immobilized on the genetic chip, and the heating temperature in the step of washing to selectively remove the similar genes having caused mismatch hybridization reaction and the unreacted adulterants. Also, commonly, any temperature variations between the respective steps have a great influence on the reproducibility of detection results.
Some means are proposed by which the detection precision and reproducibility is kept from lowering because of such temperature variations between the respective steps. For example, a polynucleotide detection chip and a polynucleotide detection system which are disclosed in Japanese Patent Application Laid-Open No. 2000-342264 employs a method in which sections are set for each kind of nucleic-acid fragments (probes) having a sequence which is complementary to the target genes contained in a specimen, and a micro-heater is provided for each section of a detecting chip so that proper temperature control can be made. In this method, the system has so greatly complicate construction that the detecting chip exclusively used therefor and also the whole detection system can not help being expensive. In addition, inasmuch as the micro heater is provided for each section, it is also difficult to make the whole system compact.
In a hybridization reaction detection method and detection system disclosed in Japanese Patent Application Laid-Open No. 2001-255328, changes with time in the course of reaction are pursued so that any influence of temperature changes of an array formed on a glass substrate can be eliminated to aim at highly precise detection. In this method, however, one-sheet array must continuously be pursued and examined using an instrument such as a microscope while the reaction is carried out, and hence it is difficult to examine a large number of arrays on substrates.
Genetic chips packaged in original shapes are also commercially available, as typified by GeneChip (available from Affymetrix Co.). These commercially available genetic chips are genetic chips supposing detection operation which utilizes a corresponding system exclusively used therefor, and hence have poor general-purpose properties. In addition, not only the system of exclusive use but also the genetic chips themselves are very expensive.
Meanwhile, in uses for researches as in universities and so forth, a genetic chip formed by immobilizing original and various detecting nucleic-acid fragments must be produced. Accordingly, a method is commonly used in which, using an array manufacturing system called a pin spotter, nucleic-acid fragments are immobilized on a slide glass substrate surface-treated with poly-L-lysine or the like. The genetic chip produced on such a general-purpose slide glass substrate, produced by this method, looks a simple glass plate in appearance. Of course, nothing is devised at all for temperature control.
Thus, it is sought to develop a detection system which can be used also when, e.g., the genetic chip is used which is formed on a general-purpose slide glass substrate, utilized in the uses for researches, has a temperature control means enabling control of temperature of the whole genetic chip in a high reproducibility and uniformity, and also has low-cost performance and high general-purpose properties as a whole.